This invention is in the area of polymeric delivery systems, and in particular is polymeric microcapsules that encapsulate gas and methods for their preparation and use.
Diagnostic ultrasound is a powerful, non-invasive tool that can be used to obtain information on the internal organs of the body. The advent of grey scale imaging and color Doppler have greatly advanced the scope and resolution of the technique. Although techniques for carrying out diagnostic ultrasound have improved significantly, there is still a need to enhance the resolution of the imaging for: (i) cardiac, solid organ, and vascular anatomic conduits (for example, the imaging of macrophage activity); (ii) solid organ perfusion; and (iii) Doppler signals of blood velocity and flow direction during real-time imaging.
Traditional, simple ultrasonic echograms reveal blood vessel walls and other echo-producing structures. However, since echoes from blood normally are not recorded, identifying which echoes are from which blood vessels is usually difficult. For example, echoes from the far wall of one blood vessel can be confused with the near wall of an adjacent blood vessel, and vice versa.
Ultrasonic contrast agents can be used to increase the amount of ultrasound reflected back to a detector. Ultrasonic contrast mediums fill the entire intraluminal space with echoes and readily permit identification of the correct pair of echoes corresponding to the walls of a particular blood vessel.
Ultrasonic contrast agents are primarily used in high-flow systems in which the contrast enhancement can be quickly evanescent. For echocardiography, a full display of bubble agents, ranging in size from two .mu.m to 12 .mu.m, and persisting from two or three to 30 seconds, has been used. For other applications, such as neurosonography, hysterosalpingography, and diagnostic procedures on solid organs, the agent must have a lifetime of more than a few circulation times and concentrate in organ systems other than the vascular tree into which it is injected. It must also be small enough to pass through the pulmonary capillary bed (less than eight microns).
Aqueous suspensions of air microbubbles are the preferred echo contrast agents due to the large differences in acoustic impedance between air and the surrounding aqueous medium. After injection into the blood stream, the air bubbles should survive at least for the duration of examination. The bubbles should be injectable intravenously and small enough to pass through the capillaries of the lungs.
The simplest suspension of air bubbles has been obtained by hand agitation of 70% dextrose or sorbitol solutions. However, this method produces large bubbles with an average diameter of greater than 15 .mu.m that exhibit a very limited in vitro stability (less than 1 minute). Feinstein, S. B., et al., J. Am. Coll. Cardiol., Vol. 3, pp. 14-20 (1984); Keller, M. W., et al., J. Ultrasound Med., Vol. 5, pp. 493-498 (1986). Smaller bubbles (usually approximately five .mu.m in diameter) have been obtained by sonicating solutions of 50% or 70% dextrose or Renografin-76 (diatrizoate meglumin 66%) but their in vitro persistence still seldom exceeds a few minutes (Feinstein, J. Am. Coll. Cardiol. 11, 59-65 (1988), and Keller, (1988)), and their in vivo persistence only a few seconds. This short lifetime may be appropriate for some applications in cardiology but may not be sufficient for organ imaging.
Air-filled particles with a polymeric shell should exhibit a longer persistence after injection than a nonpolymeric microbubble, and may be suitable not only for cardiology but also for organ and peripheral vein imaging. A variety of natural and synthetic polymers have been used to encapsulate imaging contrast agents, such as air. Research efforts in this area have to date primarily focused on agarose and alginate as the encapsulating polymers.
Agarose gel microbeads can be formed by emulsifying agarose-parafilm oil mixtures or through the use of teflon molds. In both cases, temperature-mediated gelation of agarose requires temperature elevations that render difficult the encapsulation gaseous imaging contrast agents.
Alginate, on the other hand, can be ionically cross-linked with divalent cations, in water, at room temperature, to form a hydrogel matrix, as described by Wheatley, et al., Biomaterials 11, 713-718 (1990) and Kwok, K. K., et al., Pharm. Res., Vol. 8(3) pp. 341-344 (1991). Wheatley, et al., produced ionically crosslinked microcapsules less that ten microns in diameter, where were formed of alginate, encapsulating air, for use in diagnostic ultrasound. Kwok produced microparticles in the range of 5 to 15 .mu.m by spraying a sodium alginate solution from an air-atomizing device into a calcium chloride solution to effect crosslinking, and then further crosslinking the resulting microcapsules with poly-L-lysine.
Schneider, et al., Invest. Radiol., Vol. 27, pp. 134-139 (1992) described three micron, air-filled polymeric particles. These particles were stable in plasma and under applied pressure. However, at 2.5 MHz, their echogenicity was low. Another drawback of these particles was that organic solvents (tetrahydrofuran and cyclohexane) were used to prepare the particles. Organic solvents can be difficult to remove from the microbubble and may cause a health risk to the patient.
Another type of microbubble suspension has been obtained from sonicated albumin. Feinstein, et al., J. Am. Coll. Cardiol., Vol. 11, pp. 59-65 (1988). Feinstein describes the preparation of microbubbles that are appropriately sized for transpulmonary passage with excellent stability in vitro. However, these microbubbles are short-lived in vivo (T1/2=few seconds, which is approximately equal to one circulation pass) because of their instability under pressure (Gottlieb, S., et al., J. Am. Soc. Echo, Vol. 3, pp. 328 (1990), Abstract; Shapiro, J. R., et al., J. Am. Coll. Cardiol., Vol. 16, pp. 1603-1607 (1990)).
Gelatin-encapsulated air bubbles have been described by Carroll, et al. (Carroll, B. A., et al., Invest. Radiol., Vol. 15, pp. 260-266 (1980); and Carroll, B. A., et al., Radiology, Vol. 143, pp. 747-750 (1982)), but due to their large sizes (12 and 80 .mu.m) they would likely not pass through pulmonary capillaries. Gelatin-encapsulated microbubbles have also been described in PCT/US80/00502 by Rasor Associates, Inc. These are formed by "coalescing" the gelatin.
Microbubbles stabilized by microcrystals of galactose (SHU 454 and SHU 508) have also been reported by Fritzsch, T., et al., Invest. Radiol. Vol. 23 (Suppl 1), pp. 302-305 (1988); Fritzsch, T., et al., Invest. Radiol., Vol. 25 (Suppl 1), 160-161 (1990). The microbubbles last up to 15 minutes in vitro but less than 20 seconds in vivo (Rovai, D., et al., J. Am. Coll. Cardiol., Vol. 10, pp. 125-134 (1987); Smith, M., et al., J. Am. Coll. Cardiol., Vol. 13, pp. 1622-1628 (1989).
A disadvantage of using natural polymers is that their biocompatibility is variable, and, due to impurities in the preparation extracts, it is difficult to reproduce some properties of the polymer. Synthetic polymers are preferable because they are reproducible and their properties can be tailored to specific needs, including biodegradability.
Synthetic polymers are used increasingly in medical science since they can incorporate specific properties such as strength, hydrogel characteristics, permeability and biocompatability, particularly in fields like cell encapsulation and drug delivery, where such properties are often prerequisites. However, typical methods for the fabrication of synthetic polymers into matrices or drug delivery particles involve heat, which makes encapsulating gaseous imaging contrast agents particularly difficult, or organic solvents, which may be injurious to the health of the patient.
European Patent Application No. 91810366.4 by Sintetica S. A. (0 458 745 A1) discloses air or gas microballoons bounded by an interfacially deposited polymer membrane that can be dispersed in an aqueous carrier for injection into a host animal or for oral, rectal, or urethral administration, for therapeutic or diagnostic purposes. The microballoons are prepared by the steps of: emulsifying a hydrophobic organic phase into a water phase to obtain an oil-in-water emulsion; adding to the emulsion at least one polymer in a volatile organic solvent that is insoluble in the water phase; evaporating the volatile solvent so that the polymer deposits by interfacial precipitation around the hydrophobic phase in the water suspension; and subjecting the suspension to reduced pressure to remove the hydrophobic phase and the water phase in a manner that replaces air or gas with the hydrophobic phase. There are two major disadvantages of this process. First, only polymers that have very specific solubility profiles can be used to prepare the microbubbles, i.e., they must be "interfacially depositable" on a hydrophobic phase in an aqueous medium, and soluble in a volatile organic solvent that is water-insoluble. Second, the process requires the use of organic solvents, which may be hard to completely remove from the microbubble and which may be injurious to the patient's health.
It would be useful to have a method to encapsulate imaging contrast agents with biodegradable or nonbiodegradable synthetic polymers that can be accomplished without the use of elevated temperatures or organic solvents.
Another disadvantage of current microbubble technology is the tendency of the microbubble to adhere to tissues, and the inability to effectively target the microbubbles to specific regions of interest in the body, for example, a solid tumor site or disperse tumor cells. It would be desirable to have a polymeric microbubble that has a surface that minimizes tissue adhesion, or that can be designed to target to specific regions in the body.
It is therefore an object of the present invention to provide gas-filled microcapsules made from synthetic polymers.
It is another object of the present invention to provide a gas-filled microcapsule that can persist for more than a few circulation times.
It is still another object of the present invention to provide a gas-filled microcapsule that can be prepared without the use of heat or organic solvents.
It is a further object of the present invention to provide methods for preparing these microcapsules.
It is still a further object of the present invention to provide gas-filled microcapsules that do not adhere to tissues.
It is yet a further object of the invention to provide gas-filled microcapsules that are targeted to specific regions of the body.